Abstract
Nitric oxide (NO) signaling through the formation of cGMP is well established; however, there seems to be an increasing role for cGMP-independent NO signaling. Although key molecular details remain unanswered, S-nitrosation represents an example of cGMP-independent NO signaling. This modification has garnered recent attention as it has been shown to modulate the function of several important biochemical pathways1,2,3. Although an analogy to O-phosphorylation can be drawn4, little is known about protein nitrosothiol regulation in vivo. In solution, NO readily reacts with oxygen to yield a nitrosating agent, but this process alone provides no specificity for nitrosation5. This lack of specificity is exemplified by the in vitro poly-S-nitrosation of caspase-3 (Casp-3, ref. 6) and the ryanodine receptor7. Previous in vivo work with Casp-3 suggests that a protein-assisted process may be responsible for selective S-nitrosation of the catalytic cysteine (Cys163)8. We demonstrated that a single cysteine in thioredoxin (Trx) is capable of a targeted, reversible transnitrosation reaction with Cys163 of Casp-3. A greater understanding of how S-nitrosation is mediated has broad implications for cGMP-independent signaling. The example described here also suggests a new role for Trx in the regulation of apoptosis.
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Acknowledgements
We wish to thank A. Falick (University of California, Berkeley) for acquiring the CID spectra. We also thank members of the Marletta laboratory for helpful discussions and critical review of the manuscript. This work is supported in part by grants from the DeBenedictis Fund of University of California, Berkeley to M.A.M. and US National Institutes of Health grant CA 26731.
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Supplementary information
Supplementary Fig. 1
Purification of caspase-3, wild-type thioredoxin and C73S thioredoxin. (PDF 1070 kb)
Supplementary Fig. 2
S-Nitrosation and S-glutathiolation of the catalytic nucleophile (C163) of caspase-3 by excess GSNO. (PDF 96 kb)
Supplementary Fig. 3
Poly-S-nitrosation of caspase-3. (PDF 94 kb)
Supplementary Fig. 4
S-Nitrosation of C73 of thioredoxin by excess GSNO. (PDF 55 kb)
Supplementary Fig. 5
Collision-induced dissociation spectra. (PDF 394 kb)
Supplementary Fig. 6
Caspase-3-C163-SNO S-nitrosates C73 of His-Trx. (PDF 118 kb)
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Mitchell, D., Marletta, M. Thioredoxin catalyzes the S-nitrosation of the caspase-3 active site cysteine. Nat Chem Biol 1, 154–158 (2005). https://doi.org/10.1038/nchembio720
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DOI: https://doi.org/10.1038/nchembio720
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